Multivesicular liposomes with controlled release of active agents encapsulated in the presence of a hydrochloride

ABSTRACT

Disclosed are multivesicular liposomes containing biologically active substances, and having defined size distribution, adjustable average size, adjustable internal chamber size and number, and a modulated release of the biologically active substance. The liposomes are made by a process comprising dissolving a lipid component in volatile organic solvents, adding an immiscible aqueous component containing at least one biologically active substance to be encapsulated, and adding to either or both the organic solvents and the lipid component, a hydrochloride effective to control the release rate of the biologically active substance from the multivesicular liposome. A water-in-oil emulsion is made from the two components, the emulsion is immersed into a second aqueous component, and then divided into small solvent spherules which contain even smaller aqueous chambers. The solvents arc finally removed to give an aqueous suspension of multivesicular liposomes encapsulating biologically active substances.

This application is a Divisional of U.S. Ser. No. 08/473,019, filed Jun.6, 1995, now U.S. Pat. No. 5,807,572 which is a continuation-in-part ofU.S. Ser. No. 08/352,342, filed Dec. 7, 1994, now abandoned, which is acontinuation of U.S. Ser. No. 08/020,483, filed Feb. 23, 1993, nowabandoned, which is a continuation-in-part of U.S. Ser. No. 07/709,744,filed Jun. 3, 1991, now abandoned, which is continuation-in-part of U.S.Ser. No. 07/563,365, filed Aug. 6, 1990, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/151,553, filed Feb. 18, 1988,now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the composition of syntheticmultivesicular lipid vesicles or liposomes encapsulating biologicallyactive substances and to methods for their manufacture and use.

2. Description of Related Art

Because of the easily confused acronyms, multivcsicular liposomes (MVL)have been frequently confused with multilamellar vesicles (MLV), evenamong those familiar with the art. Nevertheless, the two entities areentirely distinct from each other. Multivesicular liposomes (MVL) aredefined as liposomes containing multiple non-concentric chambers withineach liposome particle, resembling a "foam-like" matrix; whereasmultilamellar liposomes (also known as multilamellar vesicles or "MLV")contain multiple concentric chambers within each liposome particle,resembling the "layers of an onion". The distinctive structural featuresthat clearly set MVL apart from MLV are easily seen by electronmicroscopy (FIG. 1).

In addition to the above two types of liposomes, unilamellar liposomes(also known as unilamellar vesicles or "ULV") that enclose a singleinternal aqueous compartment have been described. Unilamellar liposomesinclude small unilamellar vesicles (SUV) (Huang, Biochemistry 8:334-352,1969) and large unilamellar vesicles (LUV) (Kim et al., Biochim.Biophys. Acta 646:1-10, 1981). Again, the distinctive structuralfeatures that clearly set MVL apart from unilamellar liposomes areeasily seen by electron microscopy (FIG. 1).

The prior art describes a number of techniques for producing unilamellarand multilamellar liposomes; for example, U.S. Pat. No. 4,522,803 toLenk; U.S. Pat. No. 4,310,506 to Baldeschwieler; U.S. Pat. No. 4,235,871to Papahadjopoulos; U.S. Pat. No. 4,224,179 to Schneider; U.S. Pat. No.4,078,052 to Papahadjopoulos; U.S. Pat. No. 4,394,372 to Taylor; U.S.Pat. No. 4,308,166 to Marchetti; U.S. Pat. No. 4,485,054 to Mezei; andU.S. Pat. No. 4,508,703 to Redziniak. A comprehensive review of variousmethods for preparation of unilamellar and multilamellar liposomes canbe found in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:465-508, 1980.The prior art also describes methods for producing multivesicularliposomes (Kim, et al., Biochim. Biophys. Acta 728,339-348,1983). Infact, the method of Kim, et al. (Biochim. Biophlys. Acta 728,339-348,1983) is the only report that describes multivesicular liposomes, butthe encapsulation efficiency of some of the small molecules such ascytarabine, also known as cytosine arabinoside or ara-C, was relativelylow, and the release rate of encapsulated molecules in biological fluidsat 37° C. could not be modulated. The prior-art multivesicular liposomesresult in in vivo release of the encapsulated biologically activesubstance over a period less than 24 hours following a single bolusinjection into a mammal and could not be modulated, which severelylimits the usefulness of the prior art.

The prior art by Crommelin et al. (Intl. J. Pharm. 16, 79-92, 1983)discloses that multilamellar liposomes encapsulating the biologicallyactive substance doxorubicin should be prepared in the presence of aslightly acidic medium (pH 4-6.3), because such liposomes have a slowleakage rate of doxorubicin on storage. The present invention is notintended to teach about retention or leakage of the encapsulatedbiologically substances on storage of vesicles as taught by Crommelin etal. As will be apparent from the Description of Preferred Embodiments,the purpose of the present invention is to teach modulation of releaseof the biologically active substance over time relating to its site ofapplication in the body, as anticipated by in vitro release testing inbiologically-relevant media. Furthermore, the present invention teachesthat the amount of hydrochloride added during the process ofmultivesicular liposome preparation will modulate the subsequent releaserate of the biologically active substance from the liposome at the invivo site of application. Additionally, the present invention differsfrom that disclosed by Crommelin et al. (supra) because Crommelin et al.use a distinct preparation process which results in liposomes havingentirely different structures from those of the multivesicularliposomes. Specifically, Crommelin et al. make multilamellar liposomescontaining multiple concentric chambers within each liposome particle;whereas the present art makes multivesicular liposomes containingmultiple non-concentric chambers within each liposome particle.

Optimal treatment with many drugs requires maintenance of a drug levelfor a prolonged period of time. For example, optimal anti-cancertreatment with cell cycle-specific antimetabolites requires maintenanceof a cytotoxic drug level for a prolonged period of time. Because it isa cell cycle phase-specific drug, cytarabine is a highlyschedule-dependent anti-cancer drug. Because this drug kills cells onlywhen they are making DNA, a prolonged exposure at therapeuticconcentration of the drug is required for optimal cell kill.Unfortunately, the half-life of many drugs, including cytarabine, isvery short after an intraperitoneal (IP), intravenous (IV), intrathecal(IT), intraarticular (IA), intramuscular (IM), or subcutaneous (SC)dose. To achieve optimal cancer cell kill with a cell cyclephase-specific drug like cytarabine, two major requirements need to bemet: first, the cancer must be exposed to a high concentration of thedrug without doing irreversible harm to the host; and second, the tumormust be exposed for a prolonged period of time so that all or most ofthe cancer cells have attempted to synthesize DNA in the presence of thedrug.

Prior to the present invention, it has proven difficult, costly, anddangerous to provide prolonged concentration of cytarabine in theintrathecal space. The only way of achieving a prolonged cerebrospinalfluid (CSF) drug level in the case of cytarabine has been throughcontinuous IT infusion using a drug pump. This method is not routinelyemployed because it carries a large risk of producing bacterialmeningitis. The only way of achieving a prolonged plasma drug level inthe case of cytarabine is through continuous intraveneous orsub-cutaneous infusion, both of which are inconvenient and costly. Insearching for a long-acting preparation, investigators in the past haveattempted to achieve this by chemical modification of the drug moleculeto retard metabolism or covalent attachment of a hydrophobic moiety toretard solubilization. However, such manipulations have resulted in newtoxic effects (Finkelstein, et al., Cancer Treat Rep 63:1331-1333,1979), or unacceptable pharmacokinetic or formulation problems (Ho et.al., Cancer Res. 37:1640-1643, 1977). Therefore, a slow-release depotpreparation which provides a prolonged and sustained exposure at atherapeutic concentration of a biologically-active substance is needed.The present invention is directed to the production process,composition, and use of such a preparation.

A BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A, B and C show, respectively, illustrative electron micrographsof unilamellar vesicles (ULV), which contain a single internal aqueouscompartment and usually have a diameter of 0.02-0.5 micrometers;multilamellar vesicles (MLV), which contain multiple concentric internalaqueous compartments and usually have a diameter of 0.2-5 micrometers;and multivesicular liposomes (MVL), which contain multiplenon-concentric internal aqueous compartments and usually have a diameterof 1-100 micrometers.

SUMMARY OF THE INVENTION

The present invention provides a multivesicular liposome containing abiologically active substance encapsulated in the presence of ahydrochloride, which is used to effectively modulate the release rate ofthe encapsulated biologically active substance. The multivesicularliposone system can be used to provide prolonged and sustained in vivoexposure at a disease site of a therapeutic concentration of thebiologically active substance for optimal results. The present inventionalso provides methods for making such multivesicular liposomes.

The multivesicular liposomes of this invention provide highencapsulation efficiency; controlled release rate of the encapsulatedsubstance; well defined, reproducible size distribution among theliposomes; adjustable average size that can be easily controlled; andadjustable internal chamber size and number.

The process for producing the multivesicular lipid vesicles or liposomescomprises: (a) dissolving in one or more organic solvents a lipidcomponent containing at least one neutral lipid and at least oneamphipathic lipid; (b) adding into the lipid component an immisciblefirst aqueous component containing one or more biologically activesubstances to be encapsulated; (c) adding a hydrochloride to either orboth the first aqueous component and the lipid component, forming awater-in-oil emulsion from the two immiscible components; d) dispersingthe water-in-oil emulsion with a second aqueous component to formsolvent spherules containing in them multiple droplets of the firstaqueous component, and (e) removing the organic solvents, such as byevaporation, from the solvent spherules to form the multivesicularliposomes. According to the present invention, the addition of ahydrochloride, such as hydrochloric acid or other hydrochlorides such aslysine hydrochloride, is essential for high encapsulation efficiency andcontrolled release rate of encapsulated molecules in biological fluidsand in vivo.

Accordingly, it is an object of the present invention to provide a depotpreparation which can be used to provide controlled-release of abiologically active substance at a therapeutic concentration to an invivo site.

A further object of the present invention is to provide a multivesicularliposome which can be used to provide controlled-release of abiologically active substance at a therapeutic concentration to an invivo site.

A further object of the present invention is the provision of amultivesicular liposome encapsulating a biologically active substance inthe presence of a hydrochloride which can effectively modulate therelease rate of the encapsulated biologically active substances, and canbe used to provide a controlled, prolonged and sustained exposure of thebiologically active substance at a therapeutic concentration for optimalresults.

It is a further object of the present invention to provide a method forpreparing such a depot preparation. Other and further objects, features,and advantages of the invention are inherent herein and appearthroughout the specifications and claims.

DESCRIPTION OF PREFERRED EMBODIMENTS

The term "multivesicular liposomes" as used throughout thespecifications and claims means man-made, microscopic lipid vesiclesconsisting of lipid bilayer membranes, enclosing multiple non-concentricaqueous chambers, resembling a "foam-like" matrix. In contrast,unilamellar liposomes have a single aqueous chamber; and multilamellarliposomes have a multiple "onion-skin" arrangement of concentricmembranes, in between which are aqueous compartments.

The term "solvent spherule" as used throughout the specifications andclaims means a microscopic spheroid droplet of organic solvent, withinwhich are multiple smaller droplets of aqueous solution. The solventspherules are suspended and totally immersed in a second aqueoussolution.

The term "neutral lipid" as used herein means oil or fats that have nomembrane-forming capability by themselves and lack a hydrophilic "head"group.

The term "amphipathic lipid" as used herein means a lipid that has ahydrophilic "head" group and a hydrophobic "tail" group, and hasmembrane-forming capability.

The term "anionic amphipathic lipid" as used herein means an amphipathiclipid that carries a net negative charge at pH 7.4.

The term "zwitterionic amphipathic lipid" as used herein means anamphipathic lipid that carries no net charge at pH 7.4.

The term "cationic amphipathic lipid" as used herein means anamphipathic lipid that carries a net positive charge at pH 7.4.

The term "hydrohalic acid" as used herein means a compound selected fromthe group consisting of hydrochloric acid, hydrofluoric acid,hydrobromic acid, or hydroiodic acid.

The term "hydrochloride" as used herein means hydrochloric acid or ahydrochloric acid salt of an organic base, or a combination thereof.

The term "hydrohalide" as used herein means a hydrohalic acid orhydrohalic acid salt of an organic base, or a combination thereof.

The term "modulate the release" as used herein means to increase ordecrease the release rate, or otherwise to alter the release pattern ofencapsulated biologically active substances from multivesicularliposomes.

The term "biologically active substance" as used herein means a chemicalcompound that is known in the art as having utility for modulatingbiological processes so as to achieve a desired effect in modulation ortreatment of an undesired existing condition in a living being, such asa medical, agricultural or cosmetic effect. Thus, biologically activesubstances are generally selected from the broad categories ofmedicaments, agricultural products and cosmetics. Representativebiologically active substances are disclosed in Table 1 below.

The multivesicular liposomes of the invention are made by the followingprocess, which process imparts to them their characteristic properties,including the properties of modulated release of encapsulatedbiologically active substances disclosed herein. Briefly, a"water-in-oil" emulsion containing the biologically active substance tobe encapsulated is first made by dissolving the amphipathic lipids andthe neutral lipids in a volatile organic solvent for the lipidcomponent, adding to the lipid component an immiscible first aqueouscomponent and adding a hydrochloride to either or both of the firstaqueous component and the lipid component, and then emulsifying themixture, for example, mechanically as by mixing, shaking, sonication, byultrasonic energy, nozzle atomization, or combinations thereof. Thebiologically active substance to be encapsulated can be contained in thefirst aqueous component or the lipid component, or both. The wholewater-in-oil emulsion is then mixed with the second aqueous componentand then agitated mechanically, as above, to form solvent spherulessuspended in the second aqueous component. The solvent spherules containmultiple aqueous droplets with the substance to be encapsulateddissolved therein.

The volatile organic solvent is removed, for instance by evaporation,from the spherules. When the solvent is completely evaporated,multivesicular liposomes are formed. Representative gases satisfactoryfor use in evaporating the solvent include nitrogen, helium, argon,oxygen, hydrogen and carbon dioxide. Alternatively, the organic solventcan be removed by sparging, rotary evaporation, or solvent selectivemembranes.

According to the present invention, the addition of sufficienthydrochloride effective for high encapsulation efficiency and forcontrolled release rate of encapsulated biologically active substancesin biological fluids and in vivo is essential. Hydrochloric acid is thepreferred hydrochloride, but other hydrochloridcs which are satisfactoryinclude, but are not limited to, lysine hydrochloride, histidinehydrochloride, arginine hydrochloride, triethanolamine hydrochloride,pyridine hydrochloride, and combinations thereof. Also, other hydrohalicacids, such as hydrochloric acid, hydrofluoric acid, hydrobromic acid,and hydroiodic acid, may be used. In the practice of this invention,modulation of release rate of the encapsulated biologically activesubstance is achieved by adjusting the concentration of hydrochloridepresent in either the lipid component or in the first aqueous componentused to form the water-in-oil emulsion during formation of themultivesicular liposomes to arrive at the desired release rate, i.e., atherapeutically effective release rate of a medicament or otherbiologically active substance. It should be noted that, depending uponthe concentration of hydrochloride used, the release rate of thebiologically active substance from the multivesicular liposomes caneither be increased or decreased over that achieved when thehydrochloride is not used in manufacture of the liposomes.

As one of skill in the art will appreciate, the amount of hydrochlorideused with any particular multivesicular liposome composition containinga biologically active substance will depend upon the chemical propertiesof the liposome (i.e., the combination of lipids used in itsformulation), the aqueous solution encapsulated in the liposome, theenvironment into which the liposome is to be placed (i.e., physiologicalsaline or tissue implant); as well as upon those of the biologicallyactive substance. Using the guidelines provided herein and illustratedin the Examples hereafter, one skilled in the art can readily useroutine experimentation to arrive at the amount of the hydrochloride tobe used during manufacture of the multivesicular liposome to eitherincrease or decrease the rate of release of the biologically activesubstance. For instance, as illustrated in Example 2, when the firstaqueous solution contains 20 mg/ml of cytarabine, addition ofhydrochloric acid in the range from about 10 to 70 mM during manufactureof the liposomes results in an increase in the rate of release ofcytarabine; whereas addition of hydrochloric acid in the range fromabout 70 to 500 mM results in a decrease in the rate of release of thedrug as compared with liposomes free of hydrochloric acid during theirmanufacture.

Generally, the hydrochloride and the biologically active substance areadded to the first aqueous solution if the drug or other biologicallyactive substance to be encapsulated is hydrophilic. On the other hand,if the biologically active substance is lipophilic, it is preferred toadd the biologically active component to the lipid component to be usedin formation of the water-in-oil emulsion. One skilled in the art willknow how to select the most appropriate hydrochlorides for incorporationinto either aqueous or lipid components.

Many different types of volatile hydrophobic solvents, such as ethers,halogenated ethers, hydrocarbons, esters, halogenated hydrocarbons, orFreons may be used as the lipid-phase solvent. For example, diethylether, isopropyl and other ethers, chloroform, tetrahydrofuran, ethylacetate, Forane, and combinations thereof, are satisfactory for use inmaking the multivesicular liposomes of this invention.

Various types of lipids can be used to make multivesicular liposomes,and the only two minimum requirements regarding the lipids are that thelipid component contain one neutral lipid and one amphipathic lipid.Examples of neutral lipids include diglycerides, such as diolein,dipalmitolein; propylene glycol esters such as mixed diesters ofcaprylic/capric acids on propylene glycol; triglycerides such astriolein, tripalmitolein, trilinolein, tricaprylin, and trilaurin;squalene; and combinations thereof. Examples of amphipathic lipidsinclude those with net negative charge, zero net charge, and netpositive charge at pH 7.4, such as phosphatidylglycerol (PG),cardiolipin (CL), phosphatidylserine (PS), phosphatidic acid (PA),phosphatidylinositol, phosphatidylcholine (PC), phosphatidylethanolamine(PE), sphingomyelin, diacyl trimethylammoniumpropane (DITAP), andvarious combinations thereof. Some examples of lipid combinations usedto make multivesicular liposomes include, PC/cholesterol/CL/triolein in4.5/4.5/1/1 molar ratio; PC/cholesterol/PS/triolein in 4.5/4.5/1/1 molarratio; PC/cholesterol/PG/triolein in 5/4/1/1 molar ratio;PC/phytosterol/PG/triolein in 5/4/1/1 molar ratio;PC/cholesterol/PG/tripalmitolein in 4/5/1/1 molar ratio; PG/triolein in9/1 molar ratio; PC/cholesterol/PE/triolein in 4/5/1/1 molar ratio;PE/cholesterol/CL/triolein in 4.5/4.5/1/1 molar ratio;PC/cholesterol/PA/triolein in 4.5/4.5/1/1 molar ratio; andPC/cholesterol/DITAP/triolein in 4/5/1/1 molar ratio.

Many and varied biologically active substances can be incorporated byencapsulation within the multivesicular liposomes. These include smallmolecule drugs, nucleic acids such as DNA and RNA, proteins of varioustypes including hormones produced by recombinant DNA technology,hematopoictic growth factors, monokines, lymphokines, tumor necrosisfactor, inhibin, tumor growth factor alpha and beta, cytokines,interferons, Mullerian inhibitory substance, nerve growth factor,fibroblast growth factor, platelet-derived growth factor, pituitary andhypophyseal hormones including leutenizing hormone and other releasinghormones, calcitonin, and proteins that serve as immunogens forvaccination.

Table 1 includes representative classes of biologically activesubstances which can be encapsulated in multivesicular liposomes in thepresence of a hydrochloride and which are effective in humans, andincludes biologically active substances effective for agricultural uses.

                  TABLE 1                                                         ______________________________________                                        Anesthetics  Antianginas    Antiarrhythmics                                   Antiasthmatic Agents                                                                       Antibiotics    Antidiabetics                                     Antifungals  Antihistamines Antihypertensives                                 Antiparasitics                                                                             Antineoplastics                                                                              Antivirals                                        Cardiac Glycosides                                                                         Herbicides     Hormones                                          Immunomodulators                                                                           Monoclonal Antibodies                                                                        Neurotransmitters                                 Nucleic Acids                                                                              Peptides       Pesticides                                        Proteins     Radio contrasts                                                                              Radionuclides                                     Sedatives and Analgesics                                                                   Steroids       Tranquilizers                                     Vaccines     Vasopressors                                                     ______________________________________                                    

Particularly preferred biologically active substances for use in thepractice of this invention are cytarabine, morphine, hydromorphone,leuprolide, interleukin-2, amikacin, granulocyte colony stimulatingfactor, insulin, hepatitis B vaccine, granulocyte-macrophage colonystimulating factor, methotrexate, Insulin-like Growth Factor--1, andα-interferon.

The present invention additionally provides a method of treating apathophysiological state in an individual comprising administering aliposome composition of this invention to the individual, saidcomposition comprising a biologically active substance encapsulated insaid liposome, which biologically active compound is released from theliposome at a therapeutically effective dosage rate. The term"therapeutically effective" as it pertains to the compositions of theinvention means that the biologically active therapeutic substancewithin the vesicles is released therefrom at a concentration and ratesufficient to achieve a particular medical effect for which thetherapeutic substance is intended. Examples, without limitation, ofdesirable medical effects that can be attained are chemotherapy,antibiotic therapy, and regulation of metabolism. Exact dosages willvary depending upon such factors as the particular biologically activeor therapeutic substance and desirable medical effect, as well aspatient factors such as age, sex, general condition, and the like. Thoseof skill in the art can readily take these factors into account and usethem to establish effective therapeutic concentrations without resort toundue experimentation.

Generally, however, the range of dosages of a therapeutic agentappropriate for human use in the practice of this invention includes therange of 0.1-6000 mg/sq In of bodily surface area. The reason that thisrange is so large is that for some applications, such as subcutaneousadministration, the dose required may be quite small, but for otherapplications, such as intraperitoneal administration, the dose desiredto be used may be enormous. While doses outside the foregoing dose rangemay be given, this range encompasses the breadth of use for mostbiologically active substances.

Multivesicular liposomes may be administered by any desired route; forexample, by intratumoral, intramuscular, intrathecal, intraperitoneal,subcutaneous, intravenous, intralymphatic, intraarticular, oral andsubmucosal administration, as well as by implantation (i.e., surgically)under many different kinds of epithelia, including the bronchiolarepithelia, the gastrointestinal epithelia, the urogenital epithelia, andvarious mucous membranes of the body.

The following examples illustrate the manner in which the invention canbe practiced. It is understood, however, that the examples are for thepurpose of illustration and the invention is not to be regarded aslimited to any of the specific materials and conditions therein.

EXAMPLE 1

This example illustrates a preparation of multivesicular liposomes andthe advantage of using a hydrochloride to modulate the release rate ofthe encapsulated biologically active substances, as compared to theprior art multivesicular liposomes made in the absence of ahydrochloride.

A. Preparation of Multivesicular Liposomes Encapsulating BiologicallyActive Substance in the Presence of a Hydrochloride

Step 1) In a clean one-dram glass vial (1.3 cm inner diameter×4.5 cmheight), were placed 1 ml of a chloroform solution containing 9.3 μmolesof dioleoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL),2.1 μmoles of dipalmitoyl phosphatidylglycerol (Avanti Polar Lipids), 15μmoles of cholesterol (Sigma Chemical Co., St. Louis, Mo.), 1.8 μmolesof triolein (Avanti Polar Lipids). This solution is referred to as thelipid component.

Step 2) One ml of an aqueous solution containing 20 mg/ml of cytarabine(Upjohn, Kalamazoo, Mich.) and 136 mM of hydrochloric acid, was addedinto the above one-dram glass vial containing the lipid component.

Step 3) For making the water-in-oil emulsion, the glass vial containingthe mixture of "Step 2" was sealed and attached to the head of a vortexshaker (American Scientific Products, Catalogue, McGaw Park, Ill.) andshaken at sctting "10" for 6 minutes.

Step 4) For making the solvent spherules suspended in water, thewater-in-oil emulsion obtained from "Step 3" was divided in equal volumeinto two one-dram glass vials (1.3 cm inner diameter×4.5 cm height),each containing 2.5 ml of 4 percent glucose and 40 mM lysine in water;each vial was then sealed, attached to the head of the same vortexshaker as used in "Step 3" and shaken at setting "5" for 3 seconds.

Step 5) To obtain the multivesicular liposomes, solvent spherulesuspensions produced in the two vials of "Step 4" were poured into thebottom of a 250 ml Erlenmeyer flask containing 5 ml of 3.5 g/100 mlglucose and 40 mM lysine in water. With the flask kept in 37° C. shakingwater bath, a stream of nitrogen gas at 7 L/minute was flushed throughthe flask to slowly evaporate chloroform over 10-15 minutes. Theliposomes were then isolated by centrifugation at 600×g for 5 minutes;the supernatant was decanted, and the liposome pellet was resuspended in5 ml of normal saline (0.9% sodium chloride). The liposomes wereisolated again by centrifugation at 600×g for 5 minutes. The supernatantwas decanted and the pellet was resuspended in normal saline.

B. Characterization of Multivesicular Liposomes

The mean volume-weighted diameter±standard deviation of themultivesicular liposomes obtained as described above was typically19.4±6.5 μm. The percent encapsulation of cytarabine was typically 59±7percent and the encapsulated volume typically 36±4 μl/mg of total lipidsused.

C. Preparation of Multivesicular Liposomes Encapsulating a BiologicallyActive Substance in the Absence of a Hydrochloride as a Control forComparison

Multivesicular liposomes encapsulating cytarabine in the absence ofhydrochloride were prepared according to the same procedure as describedin Part A above except that no hydrochloric acid was used in "Step 2".

D. In Vitro and In Vivo Assays for Cytarabine Release fromMultivesicular Liposomes

In Vitro assay: To a pellet of the centrifuged multivesicular liposomecontaining cytarabine, at least 100-fold volume of blood bank humanplasma containing 0.01% sodium azide was added. The liposome suspensionwas placed in a syringe and air was excluded from the syringe. Thesyringe was placed on a rotary shaker (G10 Gyrotory Shaker, NewBrunswick Scientific Co. New Brunswick, N.J.) and incubated while beingshaken at 37° C. At desired time points, an aliquot was removed from thesyringe, diluted with nonnal saline, and the multivesicular liposomeswere isolated as a pellet by centrifugation. The amount of cytarabine inmultivesicular liposomes was measured by either spectrophotometricmethod or by the method of HPLC. The percent retained cytarabine at eachtime point was calculated in reference to the drug amount at time zeroand a half-life was estimated assuming a single-exponential decay model.

In Vivo assay: Mice were injected intraperitoneally (IP) with 1 ml ofthe multivesicular liposome suspension containing cytarabine. At desiredtime points, 2 to 3 animals were sacrificed by cervical dislocation. Theperitoneal cavity was washed out thoroughly with 10 ml of normal saline.All samples were stored at -20° C. before analysis. The samples wereanalyzed for cytarabine by HPLC. The percent retained cytarabine in vivoat each time point was calculated in reference to the drug amount attime zero and a half-life was estimated assuming a single-exponentialdecay model.

Table 2 shows some representative data obtained from in vitro cytarabinerelease assay for multivesicular liposomes encapsulating cytarabine inthe presence and absence of hydrochloric acid. These data demonstratethat addition of hydrochloric acid can greatly decrease the rate ofcytarabine release out of the multivesicular liposomes. For instance,percent retained cytarabine at 24 hour was 93% when hydrochloric acidwas used, in contrast to 52% when hydrochloric acid was not used.

                  TABLE 2                                                         ______________________________________                                        In Vitro Release of Cytarabine                                                Hours of Incubation                                                                         Percent Retained Cytarabine                                     in Plasma at 37° C.                                                                  With 136 mM HCl                                                                            Without HCl                                        ______________________________________                                         0            100          100                                                 24           93           52                                                  96           78           19                                                 192           57           --                                                 336           47           --                                                 ______________________________________                                    

EXAMPLE 2

This example demonstrates that the release rate of the encapsulatedbiologically active substance from multivesicular liposomes can beeffectively modulated by varying the concentration of hydrochloric acidused.

The same procedure as described in Step 1 through Step 5 of Example 1was used for the preparation of multivesicular liposomes except with thefollowing modifications in Step 2.

Step 2) One ml of an aqueous solution containing 20 mg/ml of cytarabine(Upjohn, Kalamazoo, Mich.) and 10 to 500 mM of hydrochloric acid, wasadded into the above one-dram glass vial containing the lipid component.

Table 3 shows a summary of data obtained from in vitro cytarabinerelease assay for multivesicular liposomes encapsulating cytarabine inthe presence of various concentrations of hydrochloric acid. These datademonstrate the release rate from the multivesicular liposomes caneither be increased or decreased depending on the specific concentrationof hydrochloric acid used. For instance, when the biologically activesubstance is encapsulated in the presence of hydrochloric acid in therange of 10 to 70 mM, the release rate is increased, but when thebiologically active substance is encapsulted in the presence ofhydrochloric acid in a different concentration range, i.e. in the rangeof 70 to 500 mM hydrochloric acid, the release rate is decreased. Inother words, a method of modulating the release rate of the biologicallyactive substance by adjusting the hydrochloric acid concentration hasbeen found.

                  TABLE 3                                                         ______________________________________                                        In Vitro Release of Cytarabine                                                        Percent Retained Cytarabine                                           Hours in                                                                              at Different HCl Concentration                                        Plasma  0 mM    10 mM   20 mM 40 mM 70 mM 80 mM                               ______________________________________                                         0      100     100     100   100   100   100                                 24      59      52      39    34    15    25                                  48      52      44      39    35    20    --                                  ______________________________________                                        Hours in                                                                              90      100     200   300   400   500                                 Plasma  mM      mM      mM    mM    mM    mM                                  ______________________________________                                         0      100     100     100   100   100   100                                 24      68      86      83    86    62    68                                  48      --      65      74    82    54    78                                  ______________________________________                                    

Similarly, the in vivo release rate of cytarabine can also be modulatedby encapsulating the biologically active substance into themultivesicular liposomes in the presence of hydrochloric acid, as shownin Table 4. These latter data demonstrate that hydrochloric acid can beused to effectively reduce the release rate of cytarabine in vivo. Forinstance, addition of 100 to 400 mM of hydrochloric acid in "Step 2"during manufacture of the liposomes, as described above, resulted in asignificant increase of percent retained cytarabine at 7 and 28 hourtime points.

                  TABLE 4                                                         ______________________________________                                        In Vivo Release of Cytarabine                                                 Hours after                                                                            Percent Retained Cytarabine at                                       Injection                                                                              different HCl Concentration                                          in Mice  0 mM      70 mM     80 mM   90 mM                                    ______________________________________                                         0       100       100       100     100                                       7        2         1         5       13                                      28        0         0         0       0                                       ______________________________________                                        Hours after                                                                   Injection                                                                     in Mice  100 mM    200 mM    300 mM  400 mM                                   ______________________________________                                         0       100       100       100     100                                       7        30        33        59      35                                      28        18        25        38      7                                       ______________________________________                                    

EXAMPLE 3

This example demonstrates that the release rate of morphine frommultivesicular liposomes can be modulated by varying the concentrationof hydrochloric acid present during encapsulation of the drug into themultivesicular liposomes.

The same procedure as described in Step 1 through Step 5 of Example 1was used for the preparation of multivesicular liposomes except with thefollowing modifications in Step 2.

Step 2) One ml of an aqueous solution containing 20 mg/ml of morphinesulfate (Sigma, St. Louis, Mo.) and 0 to 300 mM of hydrochloric acid,was added into the above one-dram glass vial containing the lipidcomponent.

Table 5 shows that the release of encapsulated morphine in vitro can bemodulated by varying the concentration of hydrochloric acid used in thepreparation of multivesicular liposomes. For instance, a decreasedrelease rate of morphine was observed with an increased concentration ofhydrochloric acid in the range of 0 to 200 mM.

                  TABLE 5                                                         ______________________________________                                        In Vitro Release of Morphine                                                  Hours                                                                         in    Percent Retained Morphine at different HCl Concentration                Plasma                                                                              0 mM    25 mM   50 mM 100 mM 200 mM 300 mM                              ______________________________________                                         0    100     100     100   100    100    100                                 24    --       55     --    --     100    100                                 48     45     --       52    69    --     --                                  72    --       28     --    --      99     99                                 96     15     --       31    57    --     --                                  ______________________________________                                    

EXAMPLE 4

This example demonstrates that the release rate of iohexol frommultivesicular liposomes can be modulated by varying the concentrationof hydrochloric acid.

The same procedure as described in Step 1 through Step 5 of Example 1was used for the preparation of multivesicular liposomes except with thefollowing modifications in Step 2.

Step 2) One ml of an aqueous solution containing 20 mg/ml of iohexol(Sanofi, New York, N.Y.) and 0 to 400 mM of hydrochloric acid, was addedinto the above one-dram glass vial containing the lipid component.

Table 6 shows that the release rate of encapsulated iohexol in vitro canbe modulated by varying the concentration of hydrochloric acid used inthe preparation of multivesicular liposomes, similar to what was foundin the cases of cytarabine and morphine in examples 1-3 above. Forinstance, a slower in vitro release rate of iohexol was observed with anincreased concentration of hydrochloric acid in the range of 0 to 100mM. A further increase of hydrochloric acid concentration from 100 to400 mM resulted in a faster release rate of iohexol.

                  TABLE 6                                                         ______________________________________                                        In Vitro Release of Iohexol                                                   Hours in                                                                              Percent Retained Iohexol at different HCl Concentration               Plasma  0 mM    50 mM   100 mM  200 mM 400 mM                                 ______________________________________                                         0      100     100     100     100    100                                    24       50     105     103      68     57                                    48       54      82      86      57     55                                    ______________________________________                                    

EXAMPLE 5

This example illustrates a different procedure and a larger scale forthe preparation of multivesicular liposomes, but shows the similareffects of a hydrochloride in modulating the release of encapsulatedbiologically active substances as described in Examples 1 through 4above.

Step 1) Into a clean glass cylinder (2.5 cm inner diameter×10.0 cmheight) were placed 5 ml of a chloroform solution containing 46.5 μmolesof dioleoyl phosphatidyicholine (Avanti Polar Lipids), 10.5 μmoles ofdipalmitoyl phosphatidylglycerol (Avanti Polar Lipids), 75 μmolcs ofcholesterol (Sigma Chemical Co.), 9.0 μmoles of triolein (Avanti PolarLipids). This solution is referred to as the lipid component.

Step 2) Five ml of an aqueous solution containing 20 mg/ml of cytarabine(Upjohn, Kalamazoo, Mich.) and 0 or 136 mM of hydrochloric acid, wasadded into the above glass cylinder containing the lipid component.

Step 3) For making the water-in-oil emulsion, the mixture of "Step 2"was stirred with a TK mixer (AutoHomoMixer, Model M, Tokushu Kika,Osaka, Japan) at a speed of 9000 revolution per minute (rpm) for 8minutes.

Step 4) For making the solvent spherules suspended in water, 20 ml of asolution containing 4 percent glucose and 40 mM lysine in water waslayered on top of the water-in-oil emulsion of "Step 3" and then mixedat a speed of 4000 rpm for 60 seconds.

Step 5) To obtain the multivesicular liposomes, the solvent spherulesuspension in the glass cylinder was poured into the bottom of a 1000 mlErlenmeyer flask, containing 30 ml of 4 percent glucose and 40 mM lysinein water. With the container kept in 37° C. shaking water bath, a streamof nitrogen gas at 7 L/minute was flushed through the flask to slowlyevaporate chloroform over 20 minutes. The liposomes were then isolatedby centrifugation at 600×g for 5 minutes; the supernatant was decanted,and the liposome pellet was resuspended in 50 ml of normal saline. Theliposomes were isolated again by centrifugation at 600×g for 5 minutes.The supernatant was decanted and the pellet was resuspended in normalsaline.

EXAMPLE 6

This example demonstrates that the addition of a hydrochloride otherthan hydrochloric acid, such as arginine hydrochloride, histidinehydrochloride, lysine hydrochloride, pyridine hydrochloride, ortriethanolamine hydrochloride, in the preparation of multivesicularliposomes, is also effective in modulating the release rate of liposomeencapsulated cytarabine.

The same procedure as described in Step 1 through Step 5 of Example 1was used for the preparation of multivesicular liposomes except with thefollowing modifications in Step 1 and Step 2.

Step 1) Into a clean one-dram glass vial (1.3 cm inner diameter×4.5 cmheight), were placed 1 ml of a chloroform solution containing 13 μmolesof dioleoyl phosphatidylcholine (Avanti Polar Lipids), 2.9 μmoles ofdipalmitoyl phosphatidylglycerol (Avanti Polar Lipids), 20 μmoles ofcholesterol (Sigma Chemical Co.), 2.5 μmoles of triolein (Avanti PolarLipids). This solution is referred to as the lipid component.

Step 2) One ml of an aqueous solution containing 2 mg/ml of cytarabine(Upjohn, Kalamazoo, Mich.) and 0 or 100 mM of either argininehydrochloride, or histidine hydrochloride, or lysine hydrochloride, orpyridine hydrochloride, or triethanolamine hydrochloride, was added intothe above one-dram glass vial containing the lipid component.

Table 7 shows in vitro release data for cytarabine encapsulated inmultivesicular liposomes in the presence of either argininehydrochloride, or histidine hydrochloride, or lysine hydrochloride, orpyridine hydrochloride, or triethanolamine hydrochloride, assayedaccording to the same procedure as described in Part D of Example 1.These data demonstrate that the addition of a hydrochloric acid salt ofan organic base during encapsulation of the biologically activesubstance into the liposomes can also modulate the release rate ofcytarabine from the multivesicular liposomes incubated in plasma. Forinstance, the percent retained cytarabine at 24 hours was 95%, 91%, 97%,97%, and 62%, respectively, when arginine hydrochloride, histidinehydrochloride, lysine hydrochloride, pyridine hydrochloride, andtriethanolamine hydrochloride were used; whereas the percent retainedcytarabine at 24 hours was 50% when the hydrochloride was omitted, andreplaced with 4 percent glucose in Step 2 of the manufacturing processdisclosed above.

                  TABLE 7                                                         ______________________________________                                        In Vitro Release of Cytarabine                                                              Percent Retained Cytarabine                                     Hydrochloride   At 24 hours                                                                             At 72 hours                                         ______________________________________                                        Arginine HCl    95        117                                                 Histidine HCl   91        38                                                  Lysine HCl      97        69                                                  Pyridine HCl    97        97                                                  Triethanolamine HCl                                                                           62        33                                                  No Hydrochloride                                                                              50        10                                                  ______________________________________                                    

EXAMPLE 7

This example demonstrates that proteins can be encapsulated inmultivesicular liposomes and that a hydrochloride introduced during theencapsulation of the protein into the liposomes is effective inmodulating the release of encapsulated proteins.

The same procedure as described in Step 1 through Step 5 of Example 1was used for the preparation of multivesicular liposomes except with thefollowing modifications in Step 2.

Step 2) One ml of an aqueous solution containing 0.3 mg/ml of theprotein, granulocyte colony stimulating factor (G-CSF), (Neupogen®,Amgen Corporation, Thousand Oaks, Calif.), 175 mM glycine, and 0 or 100mM of hydrochloric acid was added into the above one-dram glass vialcontaining the lipid component.

Table 8 shows in vitro release data for G-CSF encapsulated inmultivesicular liposomes in the presence and absence of 100 mM ofhydrochloric acid, assayed according to the same procedure as describedin Part D of Example 1 except that the quantity of G-CSF was determinedby an HPLC method. These data demonstrate that addition of 100 mMhydrochloric acid reduced the release rate of G-CSF from themultivesicular liposomes when incubated in plasma. For instance, thepercent retained G-CSF at 13 and 37 hours was 51% and 24%, respectively,when 100 mM of hydrochloric acid was used; whereas the percent retainedG-CSF at 13 and 37 hours was 24% and 3%, respectively, when hydrochloricacid was omitted.

                  TABLE 8                                                         ______________________________________                                        In Vitro Release of G-CSF                                                     Hours of Incubation                                                                         Percent Retained                                                                           G-CSF                                              in Plasma at 37° C.                                                                  With 100 mM Hcl                                                                            Without HCl                                        ______________________________________                                         0            100          100                                                13            51            24                                                37            29            3                                                 84            16            0                                                 ______________________________________                                    

EXAMPLE 8

This example demonstrates that nucleic acids, such as DNA, can beencapsulated in multivesicular liposomes and that a hydrochloride iseffective in modulating the release of encapsulated nucleic acids.

The same procedure as described in Step 1 through Step 5 of Example 1was used for the preparation of multivesicular liposomes except with thefollowing modifications in Step 2.

Step 2) One ml of an aqueous solution containing 5 mg/ml of Herringsperm DNA (Sigma Chemical Co.) and 0 or 100 mM of lysine hydrochloridewas added into the above one-dram glass vial containing the lipidcomponent.

Table 9 shows in vitro release data for Herring sperm DNA encapsulatedin multivesicular liposomes in the presence and absence of 100 mM oflysine hydrochloride, assayed according to the same procedure asdescribed in Part D of Example 1. These data demonstrate that additionof 100 mM lysine hydrochloride reduced the release rate of Herring spermDNA from the multivesicular liposomes when incubated in plasma. Forinstance, the percent retained Herring sperm DNA at 24 and 120 hours was95% and 81%. respectively, when 100 mM of lysine hydrochloride was used;whereas the percent retained Herring sperm DNA was 83% and 39%,respectively, when 100 mM of lysine hydrochloride was omitted.

                  TABLE 9                                                         ______________________________________                                        In Vitro Release of Herring Sperm DNA                                         Hours of Incubation                                                                       Percent Retained DNA                                              in Plasma at 37° C.                                                                With 100 mM Lysine HCl                                                                        Without Lysine HCl                                ______________________________________                                         0          100             100                                                24         95              83                                                 72         95              40                                                120         81              39                                                192         80              38                                                ______________________________________                                    

Thus, the present invention provides methods with wide applications anduses for "depot" preparations in which biologically active substancescan be encapsulated in relatively large amounts and released at desiredrates as modulated by adjusting the hydrochloride concentration presentduring the procedure used to encapsulate the biologically activesubstance. Further, the methods of this invention allow for delivery attherapeutically effective concentrations of biologically activesubstances for optimal result while avoiding high peaking of dosage,which could often be toxic. The present invention is, therefore, wellsuited and adapted to achieve the objects as set forth in the inventionand has the advantages and features mentioned as well as others inherenttherein.

While presently preferred embodiments of the invention have been givenfor the purpose of disclosure, changes may be made therein which arewithin the spirit of the invention as defined by the scope of theappended claims.

What is claimed is:
 1. A multivesicular liposome having multiplenon-concentric chambers with membranes distributed in a matrix producedby a process comprising the steps of:(a) forming a water-in-oil emulsionfrom two immiscible components, the two immiscible components being (1)a lipid component comprising at least one organic solvent, at least oneamphipathic lipid, and a neutral lipid lacking a hydrophilic head group,and (2) a first aqueous component; said water-in-oil emulsion furthercomprising a hydrochloride selected from the group consisting ofhydrochloric acid, arginine hydrochloride, histidine hydrochloride,lysine hydrochloride and pyridine hydrochloride, and combinationsthereof, in a concentration in the range from about 10 mM to about 500mM and at least one biologically active substance, said hydrochlorideand biologically active substance being independently incorporated intoeither the lipid component or the first aqueous component, or into both;(b) dispersing the water-in-oil emulsion containing the hydrohalide intoa second aqueous component to form solvent spherules; and thereafter (c)removing the organic solvent from the solvent spherules to form themultivesicular liposomes suspended in the second aqueous component;wherein the hydrohalide concentration in the water-in-oil emulsion ischosen to modulate the in vivo release rate of the biologically activesubstance.
 2. The liposome of claim 1, wherein the hydrochloride ishydrochloric acid.
 3. The liposome of claim 1, wherein the hydrochlorideis chosen from the group consisting of lysine hydrochloride, histidinehydrochloride, arginine hydrochloride, and combinations thereof.
 4. Theliposome of claim 1, wherein the amphipathic lipid comprises at leastone zwitterionic amphipathic lipid.
 5. The liposome of claim 1, whereinthe amphipathic lipid comprises at least one cationic amphipathic lipid.6. The liposome of claim 1, wherein the amphipathic lipid comprises atleast one anionic amphipathic lipid.
 7. The liposome according to claim5 or 6, wherein the amphipathic lipid is selected from the groupconsisting of phospholipids.
 8. The liposome according to claim 7,wherein the phospholipids are selected from the group consisting ofphosphatidylcholine, cardiolipin, phosphatidylethanolamine,sphingomyelin, lysophosphatidylcholine, phosphatidylserine,phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. 9.The liposome according to claim 1 or 2, wherein the lipid componentfurther comprises cholesterol.
 10. The liposome according to claim 1 or2, wherein the lipid component further comprises a lipid selected fromthe group consisting of stearylamine, diacyl dimethylammoniumpropane,and diacyl trimethylammoniumpropane.
 11. The liposome according to claim1, or 2, wherein the biologically active substance is lipophilic and isincorporated into the lipid component.
 12. The liposome according toclaim 7, wherein the neutral lipid is a triglyceride.
 13. The liposomeaccording to claim 7, wherein the neutral lipid is a diglyceride. 14.The liposome according to claim 7, wherein the neutral lipid is apropylene glycol ester.
 15. The liposome according to claim 12, whereinthe triglyceride is selected from the group consisting of triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaproin,tricaprylin, tricaprin, trilaurin, and combinations thereof.
 16. Theliposome according to claim 13, wherein the diglyceride is selected fromthe group consisting of diolein and dipalmitolein.
 17. The liposomeaccording to claim 14, wherein the propylene glycol ester is mixeddiesters of caprylic and capric acids.
 18. The liposome according toclaim 1 or 2, wherein the organic solvent is selected from the groupcomprising ethers, hydrocarbons, esters, and combinations thereof. 19.The liposome according to claim 1 or 2, wherein the biologically activesubstance is hydrophilic and is incorporated into the first aqueouscomponent.
 20. The liposome according to claim 1 or 2, whereinemulsification of the two immiscible components is carried out using amethod selected from the group consisting of mechanical agitation,ultrasonic energy, and nozzle atomization.
 21. The liposome according toclaim 1 or 2, wherein formation of the solvent spherules is carried outusing methods selected from the group consisting of mechanicalagitation, ultrasonic energy, nozzle atomization, and combinationsthereof.
 22. The liposome according to claim 1 or 2, wherein removing ofthe organic solvent is by a method selected from the group consisting ofsparging, rotary evaporation, passing gas over the solvent spherules,and combinations thereof.
 23. The liposome according to claim 1 or 2,wherein the biologically active substance is cytarabine.
 24. Theliposome according to claim 1 or 2, wherein the biologically activesubstance is morphine.
 25. The liposome according to claim 1 or 2,wherein the biologically active substance is hydromorphone.
 26. Theliposome according to claim 1 or 2, wherein the biologically activesubstance is leuprolide.
 27. The liposome according to claim 1 or 2,wherein the biologically active substance is a nucleic acid.
 28. Theliposome according to claim 1 or 2, wherein the biologically activesubstance is interleukin-2.
 29. The liposome according to claim 1 or 2,wherein the biologically active substance is amikacin.
 30. The liposomeaccording to claim 1 or 2, wherein the biologically active substance isgranulocyte colony stimulating factor.
 31. The liposome according toclaim 1 or 2, wherein the biologically active substance is insulin. 32.The liposome according to claim 1 or 2, wherein the biologically activesubstance is hepatitis B vaccine.
 33. The liposome of claim 21, whereinthe protein is an antibody or a vaccine.
 34. The liposome of claim 21,wherein the antiparasitic is an antiviral or an antifungal.
 35. Theliposome according to claim 1 or 2, wherein the biologically activesubstance is α-interferon.
 36. The liposome according to claim 1 or 2,wherein the biologically active substance is methotrexate.
 37. Theliposome according to claim 1 or 2, wherein the biologically activesubstance is granulocyte-macrophage colony stimulating factor.
 38. Theliposome according to claim 1 or 2, wherein the lipid component furthercomprises plant sterols.
 39. The liposome according to claim 1 or 2,wherein the biologically active substance is selected from the groupconsisting of an anesthetic, an antiasthmatic agent, a cardiacglycoside, an antihypertensive, a nucleic acid, an antibiotic, avaccine, an antiarrhythmic, an antiangina, a hormone, an antidiabetic,an antineoplastic, an immunomodulator, an antifungal, a tranquilizer, asteroid, a sedative, an analgesic, a vasopressor, an antiviral, anherbicide, a pesticide, a protein, a peptide, a neurotransmitter, aradionuclide, and suitable combinations thereof.